Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

Abstract

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.

Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections

“…However, in absence of data from a considerably larger number of patients, care must be taken in interpreting the results from western blot alone, as SARS patients were found to vary in their immune responses against single proteins in western blot [14,15]. Not only western blot but also neutralisation tests should be evaluated for their capacity to afford a highly specific confirmation of serological results [7].…”

The expression of SARS–CoV M gene in P. Pastoris and the diagnostic utility of the expression product

“…Since the outbreak of SARS in 2003, several laboratory diagnostic methods have been established, including real-time RT-PCR assay, whole-virus-based immunofluorescence assay (IFA), recombinant protein-based enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests, antigencapturing enzyme-linked immunosorbent assay, and Western blot (WB) assay. These methods were critically important for controlling the transmission of SARS; furthermore, many new laboratory diagnostic methods are being developed (Poon et al, 2003(Poon et al, , 2004Shi et al, 2003;Guan et al, 2004;He et al, 2004).…”

Use of Viral Lysate Antigen Combined with Recombinant Protein in Western Immunoblot Assay as Confirmatory Test for Serodiagnosis of Severe Acute Respiratory Syndrome

“…This platform may have a similar role in our efforts to diagnose infection with SARS-CoV. In fact, recently, a few attempts have been made to utilize the Western blot platform for detecting antibodies to SARS-CoV in patients (8,19). However, these efforts mostly centered on applying a single protein marker for detection; thus, the resultant tests gave only limited information very similar to that provided by an ELISA or an immunofluorescent antibody test.…”